We are examining the dependence of EAU on specific effector cytokine profiles. Previously we demonstated that autoimmunity to retina could be either Th17 or Th1 driven and that each of these T cell responses was sufficient to mediate disease by itself. This year we have contined to study what happens when both types of effector responses are deficient. For this purpose, we generated double-deficient mice, lacking both IL-17 and IFN-&#947;, the hallmark cytokines for Th17 and Th1 responses, We find that these mice still develop EAU, but unlike wild type (WT) mice their eyes contain a mostly granulocytic inflammatory infiltrate. The double-deficient mice produce normal or enhanced quantities of IL-22, normally a part of the Th17 cytokine profile. They appear to have an enhanced Th2 response including IL-5, IL-13, an enhanced TNF-&#945;response and an unusually high IL-17F response. In vivo cytokine neutralization studies confirm that, as in wild type (WT) mice, TNF-&#945;is a pathogenic effector cytokine in this model. We are currently examining the role of IL-17F by using IL-17F deficient mice and anti-IL-17F antibodies. These results shed light on the clinical heterogeneity of human uveitis and may impact on therapy. (P. Silver and . Damsker, in preparation) We have examined the role of IL-22 in the eye. IL-22 is a cytokine typically produced by Th17 cells and is present in inflammatory sites, however, its effects on the tissue are controversial. It has been reported to have both pro-inflammatory and protective effects, depending on the site and the model. We used IL-22 and IL-22-receptor deficient mice as well as anti-IL-22 antibodies to examine effects of IL-22 modulation on EAU. Our data do not support a pro-inflammatory role for IL-22 in the eye, rather, IL-22 may have a slight protective role. (Rigden and Mattapallil, in preparation). Others have reported that it is protective in the gut and the lung. In contrast, interleukin 18 is proinflammatory in the lung. In collaboration with Peter J. M. Openshaw, National Heart and Lung Institute, Imperial College London, we demonstrated that cp-expression of IL-18 during RSV infection results in enhanced disease mediated by Natural Killer cells (ref. 1). The innate immune response directly affects immunopathogenic processes and also impacts on adaptive immunity. We previously identified a population of NKT cells that are NK1.1 and rapidly produce IL-17 independently of IL-6 and IL-21. We have now identified a novel population of innate T cells, which are distinct from other innate T cells including the NKT cells mentioned above. These cells have an antigen (Ag) experienced phenotype, lack both CD4 and CD8 expression (double negative = DNT) and rapidly produce high amounts of IL-17 upon T cell receptor and IL-23 receptor ligation, but do not produce significant amounts of IFN-&#947;. Similarly to NKT cells, production of IL-17 by these cells is independent of IL-6 and IL-21, which we showed using the appropriate cytokine deficient mice. We are currently characterizing this cell population with the goal of defining its role in host defense and tissue pathology (A. Hansen, in preparation). In collaboration with Drs. Warren Strober (NIAID) and Guanxun Meng (now at Shanghai Pasteur Institute) we are studying uveitis in mice with an inflammasome mutation affecting NLRP3. These mice are knock-in for a Cryopyrin-associated periodic syndromes (CAPS) mutation and constitue a model for Blau syndrome. Preliminary results show some effects on immune responses and histology of innate (LPS) as well as autoimmune-driven (EAU) ocular inflammations. We have developed T cell receptor (TCR) Tg mice carrying a TCR specific to the major epitope of IRBP (IRBP TCR Tg). On a conventional background, these mice express of 20-25% of IRBP-specific T cells, as detected by a specific peptide-MHC class II-Ig dimer developed in our lab, and almost 100% specific T cells on a RAG2-/- background. They develop spontaneous EAU disease by 2 months of age, that can be very severe. These mice represent a new model of spontaneous EAU and are serving as a tool to study the development, migration and function of antigen-specific cells in uveitis. In addition, they serve as donors of na&#239;ve Ag specific T cells for various studies (see ahead). Interestingly, spontaneous uveitis is ameliorated if the IRBP TCR Tg mice are treated with a broad spectrum antibiotic mixture from before birth, resulting in a drastically altered gut bactreial flora. This suggests that endogenous bacterial flora contributes to development of ocular autoimmune disease. The findings will be confirmed in germ-free mice (Horai et al, manuscript in preparation). Stimulation through Toll receptors (TLRs) and other innate receptors may contribute to this. In collaboration with Dr. Shao Bo Su and his group at the zhongshan Eye Center, Sun Yat Sen University in Guangzhu, China, we found that in mice immunized with a low-dose EAU regimen, TLR4 agonist lipopolysaccharide (LPS) as well as agonists TLR2, TRL3, or TRL9 enhanced EAU scores and the associated immunological responses (ref. 2). The internal microenvironment within the eye is immunoinhibitory, a property that is part of the immune privilege of the eye and is believed to protect they eye from inflammation. In vitro data demonstrated that TGF-&#946;and other substances in the ocular fluids, as well as contact with resident ocular cells, can inhibit T cells and even convert them to T-regulatory (Treg) cells. However, proof that this can happen in vivo was lacking. Using IRBP specific cells from T cell receptor transgenic mice (above) we demonstrated that na&#239;ve conventional T cells convert to FoxP3+ T-regulatory cells in the living eye in a process requiring local antigen recognition, and we studied the mechanisms at the molecular level. We further demonstrated that retinoic acid is necessary for the conversion despite high levels of TGF-&#946;present in the eye, and thus plays a dual role: in vision and in ocular immune privilege. Finally, our data reveal that na&#239;ve T cells can be primed within the target tissue itself, rather than only in lymph nodes. Nevertheless, differentiated effector cells are relatively insensitive to AH, helping to explain occurrence of uveitis in the face of an inhibitory ocular microenvironment. These findings shed new light on the phenomenon of immune privilege and on its role in actively controlling immune responses in the tissue. (Ru Zhou, submitted) Immunological responses to S-Ag have been implicated in human uveitis, however, direct study of uveitogenic epitopes in humans is not possible. We have established a "humanized" EAU model in HLA transgenic (Tg) mice. Using bioinformatic methods for epitope prediction we are studying recognition and pathogenicity of S-Ag epitopes, and identifying their core sequences. We have identified permissive HLA-DR3(0301), HLA-DR4(0402), HLA-DQ8 and non-permissive HLA-DR4(0401), HLA-DR2 (*1501, *1502, and *1503) alleles of the HLA-DR and DQ genes, and characterized some of the allele-specific epitopes of human S-Ag. Importantly, the sequences of these epitopes overlap with peptides M and N of S-Ag, and the S-Ag crossreactive B27PD peptide derived from HLA-B27 molecule, that were reported to be recognized by lymphocytes of uveitis patients. We have developed MHC tetramers loaded with these peptides to study S-Ag specific lymphocytes and have demonstrated that they can detect antigen-specific cells in HLA Tg mice with EAU. We are using these tetramers to detect antigen-specific cells in uveitis patients of the appropriate HLA types, with the goal of developing this method into a diagnostic tool and biomarker for antigen-specific responses in patients (Mattapallil et al., in preparation).